Members of the transferrin family (serum transferrin, ovotransferrin &lactoferrin) bind iron with different affinities in each of two lobes, although all lobes have identical amino acid ligands to the iron. Iron sequestration and transport by serum transferrin and delivery of this iron to cells by pH dependant, receptor mediated endocytosis, are biologically critical functions. At the core of this proposal is our desire to mechanistically understand iron release from the individual lobes of human serum transferrin and ovotransferrin, especially in a complex with their respective receptors. We believe that both lobes of transferrin interact with each other and with the receptor to bring about release of this vital metal. Key to the proposed studies is our ability to make site-directed mutants for which spectral properties (UV-vis and EPR) and the rate constants for iron release in the presence and absence of our recombinant soluble transferrin receptor (as a function of pH and anion concentration) can be measured. Authentic monoferric and apo- transferrins provide essential controls. The conformational dependence on recognition of the mutants by the receptor will be initially assessed by surface plasmon resonance (SPR) measurements to obtain affinity constants as a function of pH and iron saturation. Some samples will be evaluated by isothermal titration calorimetry (ITC) to provide thermodynamic parameters to more precisely evaluate the interactions. Equilibrium binding studies and cellular uptake experiments will allow further assessment of the interaction between transferrin and the receptor and show whether a particular mutant retains the ability to donate iron to cells. This cell work provides essential in vivo data to support our in vitro studies. Integral to our studies will be structure determinations of human serum transferrin and some of our mutants allowing us to link structure to function. Overall we wish to precisely identify the steps leading to iron release by identification of the specific residues within each lobe of transferrin that facilitate the efficient delivery of iron at the right time in the right place. The interaction of TF with its specific receptor controls iron distribution in the body. Owing to the fact that iron deficiency and excess are directly related to specific human diseases, understanding this process at the molecular level is essential to a global understanding of iron metabolism.